Fig. 7

Dexamethasone and rapamycin still exerted synergistic cytoprotection with post-treatment after challenge of cytokines in A549 cells. a A549 was stimulated with the cytokine mixture (CM). Co-treatment of dexamethasone (D, 10 nM) and rapamycin(R, 1 nM) was initiated 1h before (− 1 h), 1 h after (+ 1 h), 2 h after (+ 2 h), 4 h after (+ 4 h), 7 h after (+ 7 h) cytokine stimulation, and released LDH was evaluated 48 h after cytokine stimulation as described in the “Methods” section. Post-treatment of dexamethasone and rapamycin still inhibited LDH release significantly at least up to 7 h after cytokine challenge, although the degree of cytoprotection gradually decreased compared to pre-treatment (− 1 h). Bar graph shows means ± SEM. *P < 0.01 vs. CM alone. b A549 cells were treated with ethanol (vehicle) or dexamethasone (D, 10 nM), rapamycin (R, 1 nM) or both (DR) 2 h after cytokine stimulation. Forty-eight hours after cytokine stimulation, cytotoxicity (grey bar graph) and apoptosis (black bar graph) were evaluated as described in the “Methods” section. Bar graph shows means ± SEM. *P < 0.01 vs. CM alone, **P < 0.01 vs. CM/D, CM/R. c A549 cells were treated with ethanol (vehicle) or dexamethasone (D, 10 nM), rapamycin (R, 1 nM) or both (DR) 2 h after CM stimulation. Total cell lysates were collected 48 h after CM stimulation and then subjected to western blotting using the indicated antibody. Both of augmentation of phosphorylated Akt(p-Akt) and inhibition of phosphorylated c-Jun(p-c-Jun) were still remarkable in post-treatment of both of dexamethasone and rapamycin compared with either dexamethasone or rapamycin treatment